The continually dropping costs of sequencing have opened the possibilities of clinically using DNA sequencing, apart from the traditional field of clinical genetics, in oncology as well as in other fields like plant breeding, pathogen detection as well as fields of research.
At the moment most commercial instruments for sequencing and the ones in development as next generation have an only partly automated procedure. The detection of the bases in each strand is performed and the resulting reads of the DNA are outputted. Largely, the sample preparation before the sequencing procedure itself is still performed by trained personnel. In this way significant time and resources are being spent. The time line of a present sequencing run spans usually over three weeks, of which one week is the sample preparation, another week is the automated detection of bases of DNA strands and one week is needed for bio-informatics analysis.
After the extraction of the DNA from the cells the molecules have to be fragmented before the detection of the bases starts to take place on the same cartridge. It is preferable that the average size of the DNA strands is approximately 200 base pairs (bp). This particular length is equal with the ideal read length required in the computational analysis. It is clear that the minimum amount of required DNA material (from biopsies for instance) can be reduced significantly if the fragmentation procedure leads to average size of the molecules LAVG that is as close as possible to the ideal read length. Furthermore it is preferable that LAVG can be tuned to a certain length depending on the minimum requirements of the following steps.
It should be noted that the commonly accepted method for selecting DNA fragments with proper length consists in the following steps: a) perform a given fragmentation method, b) run a gel electrophoresis experiment where a calibration ladder is placed, c) select the fragments at the proper length by cutting out the corresponding part of the gel. Alternatively an automated instrument can perform a similar operation with more reproducible results. Such device requires trained personnel and approximately one hour of processing.
EP 0 353 365 describes an ultrasonic cell-destroyer in which different solution containers are provided in a closure plate 21. US 2002/0039783 A1 describes a device for lysing cells, spores, or microorganisms. US 2002/0187547 A1 describes a container for holding cells for disruption.
From WO 2011048521 A1 microfluidic cartridge with a parallel pneumatic interface plate is known. Such a device makes it possible to move a liquid a long predefined path on a microfluidic cartridge.
Although there are several methods for DNA fragmentation only a few can be considered adequate for special purposes. However, all of them seem to have at least one disadvantage which needs to be improved. A few of these methods are mentioned herein. Firstly the enzymatic digestion is a method that is used in DNA fragmentation. The disadvantage of this method is that the places where the strands are cut are not randomly distributed. This can be a source of errors in the computational analysis. Secondly flow boundary layer method is a fluidic method for fragmentation. This method requires substantial pressures to be developed on the cartridge. The fluid containing the DNA molecules is passed through an orifice at very high flow rates Q on the order of hundreds of ml/min. For instance it has been reported that for LAVG=1000 bp, Q=125 ml/min is required. The orifice used had the length L=1 mm and radius R=125 μm. The pressure difference developed in these conditions would have been
  p  =                              8          ⁢          L          ⁢                                          ⁢          η                          π          ⁢                                          ⁢                      R            4                              ⁢      Q        =          350      ⁢                          ⁢              MPa        .            It is clear that such method integrated in a cartridge would require unacceptable costs of fabrication due to the components to be used, e.g. shearing orifice made of ruby. Thirdly sonication is another method that can be successfully used for shearing DNA molecules. Indeed the method can have LAVG˜200 bp. It consists of immersing an ultrasound transducer or an acoustical wave guide into the sample fluid. Such procedure brings the risk of sample contamination and the minimum volumes to be used are in the order of hundreds of micro-liters. In addition the risk of foaming the sample is substantial when some buffers are used. Foaming can be easily obtained when trying to re-disperse beads sediment after long storage time. Also, sonication is not a solution for a sequencing system with a closed disposable cartridge. Fourthly the use of the principles of ultrasonic cavitation is known. Acoustic pressure fields are applied in order to trigger bubble nucleation followed by cavitation. As a result pressure gradients are created along the DNA molecules resulting in fragmentation. The sample is placed in a closed container located in the focal region of an ultrasound transducer. Known devices using such cavitation can be used for sample volumes ranging from 50 μl to milliliters. Furthermore the known devices have active cooling to control the water bath in which the transducer is placed. A large water container (>25 cm×25 cm×25 cm) is required to accommodate the ultrasound transducer, the cooling circuit and the sample fixture. Due to its dimensions such device would be difficult or impossible to integrate in a sample-preparation station. Fifthly wind stress boundary layer and boundary-induced acoustic streaming is known for fragmentation. In this case the sample is placed in the proximity of an ultrasound transducer. In case solid material is present in the propagation path of ultrasound an inherent increase of the transducer temperature during a treatment and other physical parameters may occur. Therefore the impedance properties of the solid material may change from experiment to experiment leading to serious reproducibility issues.